SupportLigon Research Center of Vision at Kresge Eye Institute, Dryer Foundation, NIH Core Grant EY04068 to Department of Anatomy and Cell Biology at Wayne State University, and Research to Prevent Blindness to Department of Ophthalmology at Wayne State University.

Purpose :
Channelrhodopsin (ChR), such as ChR2, based optogenetics is one of the promising approaches to restore vision in photoreceptor degenerative diseases, such as retinitis pigmentosa. The development of highly light-sensitive ChRs that can restore functional vision under normal light conditions is desired. We have previously reported a strategy of improving light-sensitivity of ChR2 by optimizing its kinetics (Pan et al., 2014). Recently, a new ChR variant from Chloromonas oogama, CoChR, has been shown to exhibit larger photo-current than ChR2 (Klapoetke et al., 2014). The aim of this study is to develop more light-sensitive CoChR mutants for optogenetic vision restoration.

Methods :
CoChR mutants (CoChRms) were created by site-directed mutagenesis. All CoChRs were fused with GFP and cloned into a mammalian expression vector with CMV or CAG promoter. The expression and light response properties were evaluated in HEK cells by patch-clamp recordings. ChRs in the retina was delivered by AAV2 vectors via intravitreal administration. Restoration of vision was assessed by optomotor behavior in a triple knock-out (TKO; Gnat1-/-Cnga3-/-Opn4-/-) mouse model using a LED-based homemade optomotor system or a computer-based virtual optomotor system (Prusky et al., 2004).

Results :
The photocurrent of wt-CoChR shows a peak sensitive wavelength of 480 nm and a relatively fast off-rate of 112 ± 35.6 ms. We created several more light-sensitive CoChRms by increasing the off-rate and/or improving expression. The most light-sensitive CoChR mutant, CoChR-H94E/L112C/K264T (CoChR3m), showed an off-rate of 635 ± 109 ms. Its peak photocurrent (872 ± 289 pA) evoked at a low light stimulation was over 2-fold higher than that of wt-CoChR (368 ± 140 pA). In addition, its current desensitization (12%) was markedly reduced compared to that of wt-CoChR (60%). Optomotor responses for CoChR3m-treated mice were observed at the light intensity of ~1 x 1013 photons/cm2s with a wavelength of ~470 nm. Furthermore, optomotor responses could also be elicited by using the virtual optomotor system. Visual acuity up to 0.16 cycles/degree (c/d) and contrast sensitivity up to 4 (25%) at the spatial frequency of 0.042 c/d were observed.

Conclusions :
We developed an ultra light-sensitive CoChR mutant which enables to restore functional vision in a blind mouse model under ambient light conditions.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.